As a response to the rapid increase in available genetic information and its impact on molecular biology, health care, treatment modalities, pharmaceutical research, epidemiological studies, etc., the scientific interest is today focusing on the cellular effects of the genetical key elements as well as their biological role and functions. As the accumulation of new information related to the basic key elements in genetics is slowing down, the desire to study their effects and/or biological role is steadily increasing.
Bioinformatics, handling information related to life processes and accumulating in genomics, proteomics, transcriptomics, etc., in a mathematically exact manner has created a demand for new accurate tools allowing rapid and quantitative assessment of the effects and importance of the accumulated knowledge. Combinatorial chemistry allows rapid synthesis of an enormous amount of novel and already existing compounds. It is desirable to rapidly assess the effects and potential importance of said novel or already existing compounds or other external stimuli on the gene expression in living organisms, including human beings and experimental animals. In other words, the information accumulating in genomics, proteomics, transcriptomics, etc., as well as combinatorial chemistry combined with bioinformatics has created a demand for new tools allowing rapid, accurate and preferably quantitative assessment of the effects and biological role of said compounds. In fact, a significant market has grown up around the technology allowing transcriptional profiling. Transcriptional profiles are not only used by scientists in many areas of basic research in life sciences, but also in industrial research and development. The effects of known and novel drugs on the gene expression of human beings and experimental animals is today an essential knowledge in the pharmaceutical and diagnostic industry, but beneficiaries will also be several other sectors of the biotechnology industry.
A powerful tool in transcriptional profiling is the oligomer-chip technology, disclosed for example in the following U.S. Pat. No. 6,040,138, U.S. Pat. No. 5,556,752; U.S. Pat. No. 5,770,722, U.S. Pat. No. 5,807,522 or patent application WO 200003037. The U.S. Pat. No. 6,040,138 discloses the simultaneous monitoring of multiple gene expression by hybridizing target nucleic acids to an array of immobilized oligonucleotide probes. The U.S. Pat. No. 5,556,752 and U.S. Pat. No. 5,770,722 describe nucleic acid sequencing and methods of analysing by the aid of nucleic acid library arrays on solid supports as well as by applying hybridisation and nuclease or ligase reactions. In patent application WO 200003037 the screening of target polynucleotides on an array to determine their genetic function is disclosed. U.S. Pat. No. 5,807,522 discloses micro-arrays of certain analyte-assay regions containing analyte-specific reagents, which are useful for many genetic applications applying large-scale hybridisation techniques.
The common characteristic of the microarray techniques described above is that the probes, i.e. the polynucleotide sequences used as reagents are immobilized or coupled to a solid carrier. The immobilization of the probes acts as a steric hindrance and prevents the hybridization to take place in a stochiometric fashion resulting in low yield. Thus, the methods mentioned above are only semiquantitative and require double labeling and comparative checking.
Due to insufficient distinguishability caused, e.g. by blurred probe spots, it is often impossible to compare the spots with sufficient accuracy. Although this is not an obstacle for applying the micro-array techniques, it remains a problem, when quantitative results are needed and it explains why a large degree of redundancy is required in the actual tests. In the patent application WO 98/51789 the preparation of subdivided cDNA libraries from mRNA by reverse transcription and amplification is described. The libraries are used for screening new genes, interacting proteins, potential drugs and/or for diagnosing. This system depends on PCR-technology and a set of different primers. Even if said system allows detection of down-regulated sequences present in low amounts, it increases quantification problems. Accordingly, powerful tools for studying the biological role of genetical key elements exist, but the problem of obtaining quantitative results still remains. Furthermore, the previous techniques are not applicable for monitoring the effects of uncharacterized genomes.
Thus, the main objective of the present invention is to provide a method and test kits not only for quantification of expression patterns or transcriptional profiles enabling comparative assessments of variations therein, but also to provide a very sensitive test, which allows the quantitative determination of very small amounts of analyte polynucleotides, which otherwise would be under the detection limit. The objective is to provide a truly quantitative method and test kit, which as an answer gives the amount of polynucleotide copies in the sample, e.g. allows the number of mRNA copies present in the sample to be assessed and which further can be modified to increase its sensitivity significantly.
An advantage of the method and test kit of the present invention is that it allows assessment of transcriptional profiles or expression patterns not only for characterized but also for uncharacterized genomes.
A further advantage of the present invention is that the quality of the analyte, i.e. the polynucleotide preparation, to be analyzed, is not critical. RNA, which generally, is known to require special treatment due to its instability, can be used directly for the quantitative assessment.
The manufacturing of test kits, which need not include immobilization steps and certain commercially available reagents allows easy adaptation of tailor-made tests, directing the attention to certain subsets of genes in a given organism.
The method is very adaptable. It can be used in fully automatic or semiautomatic assemblies. The procedure can be interrupted at several stages. The samples and reaction products can be preserved until sufficient data has been collected or it is more convenient to continue the process, e.g. recording the results.